B cell lineage reconstitution underlies CAR-T cell therapeutic efficacy in patients with refractory myasthenia gravis

B-cell maturation antigen (BCMA), expressed in plasmablasts and plasma cells, could serve as a promising therapeutic target for autoimmune diseases. We reported here chimeric antigen receptor (CAR) T cells targeting BCMA in two patients with highly relapsed and refractory myasthenia gravis (one with AChR-IgG, and one with MuSk-IgG). Both patients exhibited favorable safety profiles and persistent clinical improvements over 18 months. Reconstitution of B-cell lineages with sustained reduced pathogenic autoantibodies might underlie the therapeutic efficacy. To identify the possible mechanisms underlying the therapeutic efficacy of CAR-T cells in these patients, longitudinal single-cell RNA and TCR sequencing was conducted on serial blood samples post infusion as well as their matching infusion products. By tracking the temporal evolution of CAR-T phenotypes, we demonstrated that proliferating cytotoxic-like CD8 clones were the main effectors in autoimmunity, whereas compromised cytotoxic and proliferation signature and profound mitochondrial dysfunction in CD8+ Te cells before infusion and subsequently defect CAR-T cells after manufacture might explain their characteristics in these patients. Our findings may guide future studies to improve CAR T-cell immunotherapy in autoimmune diseases.

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EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection.Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status.***** Reviewer's comments ***** Referee #1 (Remarks for Author): BCMA-directed CAR-R treatment has already been applied to treat multiple myeloma, and another RNA-CAR-T therapy has been tested for refractory MG that did not use DNA vector or require lymphodepletion in patients.Merit: 1.The current investigation represents another approach to test the clinical efficacy of the CAR-T treatment using DNA vectors and refractory MG patients.2. The manuscript is well written, and the data presented in the manuscript are clear and sufficiently described.3. The characterization of engineered cells after transfer and the RNA-seq analysis of targeted cells are all logical approaches.There are some limitations of the study: 1.The study involved only one participant for each MG-type (AChR-IgG and MUsK-IgG), which affects the reliability of the results due to lack of statistical power.The results from one patient cannot be generalized for all MG patients.It undermines the purpose of the study, and any firm conclusions cannot be derived.2. The study participants, who were refractory to prednisone, tacrolimus, and rituximab, discontinued the treatments only 1-3 days before starting CAR-T therapy.Thus, it is difficult to determine whether the clinical benefits observed in the two patients solely resulted from the CAR-T treatments or partly as an outcome from the previous treatments.3. The authors found that the CAR-T-anti-BCMA treatment in patients reconstituted B cell lineages with naïve phenotypes.They need to clarify that statement as BCMA is present only on plasmablasts and plasma cells, but not on precursor B cells that differentiate to plasma cells.
Referee #2 (Comments on Novelty/Model System for Author): The quality of the methodology is high as well as the importance of the research question.Since this is not the first study of these characteristics in MG I see the impact as medium.additionally, I see the translation to the clinic low in its present form since there are other available treatment alternative which are less invasive.

Referee #2 (Remarks for Author):
In this manuscript, the authors claim that CAR-BCMA T cells are well-tolerated and highly effective in treating refractory MG, resulting in an improvement in physical function and serologic remission.Patient MG-1, with anti-AChR-IgG and anti-Titin antibodies, and patient MG-2, with anti-MuSK-IgG4, showed a poor response to first-line immunosuppressive therapies.Although B-cell-targeting therapy rituximab was considered an early therapeutic option, it also failed in these two patients.The present case series illustrates the potential of CAR-T cell therapy in treating seropositive MG, as evidenced by sustained depletion of autoantibodies and prolonged therapeutic efficacy beyond one year.
The reviewer has concerns regarding the choice of applying CAR T-cell therapy to these patients, as there are other clear options available with lower potential side effects, specifically targeting plasma cells and the effector functions of anti-AChR antibodies.Refer to Mané-Damas, Marina et al. "Novel treatment strategies for acetylcholine receptor antibody-positive myasthenia gravis and related disorders" (Autoimmunity Reviews, vol. 21,7, 2022, 103104. doi:10.1016/j.autrev.2022.103104).
As is well known, CAR T-cell therapy is a type of immunotherapy that involves genetically modifying a patient's T cells to express a receptor targeting cancer cells.Neurological side effects, particularly neurotoxicity or cytokine release syndrome (CRS), can occur as a result of this therapy.The onset and duration of neurological side effects can vary widely among individuals.Neurological side effects from CAR T-cell therapy typically manifest within the first few days to weeks after treatment initiation.In some cases, they can occur within hours after the infusion of modified T cells.The duration of these side effects also varies.Some individuals may experience transient and mild symptoms, while others may face more severe and prolonged effects.The severity and timing of neurological side effects are often unpredictable.Symptoms may include confusion, delirium, seizures, and other neurological manifestations.Patients receiving CAR T-cell therapy should have regular follow-up appointments with their healthcare team to monitor for any delayed or long-term side effects.
It is crucial to consider if the potential side effects of this treatment are worthwhile, especially in neurological diseases like MG, when other treatment options are available in the market.
I would not consider such a complex approach, as depicted here, for the treatment of refractory MG patients a good current option.Instead, I would seek, if possible, more specific patient-tailored strategies, which will minimize the considerable risk of side effects, especially in the long term.
Referee #3 (Remarks for Author): In this manuscript, Tian and colleagues reported very favorable outcome using chimeric antigen receptor (CAR) T cells targeting BCMA in two patients with refractory myasthenia gravis (one with AChR Ab, and one with MuSk Ab).They also utilized single cell RNA and TCR sequencing to track the temporal evolution of CAR-T phenotypes, and found a shift from autologous T effector (Te) to proliferating cytotoxic-like CD8 clones and NK-like Te cells post treatment in vivo.The clinical outcome and decline in antibody titers at 12 mo are significant and expected, but whether long-term remission will be induced remains to be determined.The work is exciting and very thorough and provides insight into immunologic events occurring after CAR-T cell therapy targeting BCMA in autoimmunity.Data presentation is excellent though may be too crowded /complex in the last 2 figures.Data interpretations are logical.Here are some questions /comments: 1. Cyclophosphamide has occasionally been used in refractory MG.Can the authors comment on the possibility that lymphodepletion prior to CAR-T cell infusion may have partially contributed to favorable outcome? 2. Since the number of targeted cells (BCMA+ B cells) in autoimmune diseases is much lower than in lymphomas or other cancers, is lymphodepletion allowing favorable condition for CAR-T cell proliferation necessary?3. Can authors explain why do CAR-T cells persist only 30d in autoimmune disease compared to longer persistence in cancers? 4. In Fig. 1D on inflammatory mediators, do data represent transcript or protein levels? 5.In Fig. 3D legend, "The line width is proportional to the communication probability in NMOSD comparing to control group".I assume that the authors meant MG and not NMOSD.6. Changes in MIF and serum BCMA levels have the potential to be used as biomarkers of response to therapy.Do authors have data on MIF and serum BCMA at 12 months post CAR-T cell therapy? 7. Fig. 4C legend can be expanded to indicate lower proliferation and energy metabolism and higher cytotoxicity at 1 mo compared with CAR-T cells in IP. 8. Mitochondrial dysfunction/oxidative phosphorylation abnormalities have been reported in SLE B lymphocytes (Takeshima et al 2022).Thus, impaired mitochondrial function in Te in this study may not be specific to myasthenia, and effect of prolonged exposure to immunosuppressants may be a contributing factor.
There are some limitations of the study: 1.The study involved only one participant for each MG-type (AChR-IgG and MUsK-IgG), which affects the reliability of the results due to lack of statistical power.The results from one patient cannot be generalized for all MG patients.It undermines the purpose of the study, and any firm conclusions cannot be derived.Response: We thank the reviewer for pointing out the limitations of our study.Indeed, we only reported here information on two patients before and after receiving CAR-T treatment.Although the number of examples is small, they have a strong representative significance.The patient MG-1 had typical AChR-IgG-positive myasthenia gravis.Her symptoms were not controlled under treatment with acetylcholinesterase inhibitor, prednisolone, and tacrolimus or rituximab.She had recurring crises before treatment and was unable to take care of herself.Another patient (MG-2) had typical MuSK-IgG-positive myasthenia gravis.Before enrolment, she had obvious symptoms in bulbar muscles, showing pharyngeal, and tongue weakness.After receiving CAR-T treatment, these two patients completely achieved drug-free clinical remission during a follow-up period of up to 18 months (our current follow-up period has been extended to 18 months, and these two patients completely stopped treatment with steroids, and all kinds of immunosuppressants).Currently only pyridostigmine is used, 30 mg twice a day for maintenance.These two cases mainly demonstrate the potentials of anti-BMCA CAR-T therapy in the two classic subtypes of myasthenia gravis.We must admit that ONLY two patients suggesting that the prospects of CAR-T therapy have certain limitations, and we have now added corresponding descriptions in the limitations (Page 13 Line 512-515 in marked version).Thanks again for the constructive suggestions.
2. The study participants, who were refractory to prednisone, tacrolimus, and rituximab, discontinued the treatments only 1-3 days before starting CAR-T therapy.Thus, it is difficult to determine whether the clinical benefits observed in the two patients solely resulted from the 20th Dec 2023 1st Authors' Response to Reviewers CAR-T treatments or partly as an outcome from the previous treatments.Response: First, we thank the reviewer for pointing out this issue.We are very sorry that the lack of detailed description has caused some misunderstandings.Although both patients failed traditional treatment, the risk of recurrence or worsening that may arise from longer drug withdrawal is unethical.So, we only stopped oral steroids and immunosuppressants 1-3 days before lymphodepletion treatment.In the revised manuscript, we describe in detail the time points at which patients receive different treatments before CAR-T infusion and add related information to Figure 1.At the same time, both patients have been followed up for more than 18 months.We have also expanded the clinical follow-up data to 18 months and added it to the results and Revised Figure 1 Revised Figure 2 3.The authors found that the CAR-T-anti-BCMA treatment in patients reconstituted B cell lineages with naïve phenotypes.They need to clarify that statement as BCMA is present only on plasmablasts and plasma cells, but not on precursor B cells that differentiate to plasma cells.Response: Thank you very much to the reviewer for such professional suggestions.We speculate that the anti-BCMA CAR-T cells could remove long-lived plasma cells and plasmablasts that secrete autoantibodies, and then the B cells that grow up again after lymphodepletion treatment do not differentiate into pathogenic plasma cells and plasmablasts.However, the mechanism underlying the reconstitution of B cell lineage is currently unknown and needs further exploration.We have added related description of the distribution of BCMA to the discussion (Page 12 Line 462-468 in marked version) and the reconstitution of B cells after anti-BCMA CAR-T cell therapy.Thank you again for all the constructive suggestions.
Referee #2 (Comments on Novelty/Model System for Author): The quality of the methodology is high as well as the importance of the research question.Since this is not the first study of these characteristics in MG I see the impact as medium.additionally, I see the translation to the clinic low in its present form since there are other available treatment alternative which are less invasive.Response: We are very grateful to the reviewer for pointing out the importance of our research.Regarding the comparison between our report and other articles on CAR-T treatment of MG, as well as the comparison with other "less invasive" treatments, we carefully summarized the relevant research progress and added a lot of comments in the discussion.Many thanks to the reviewer for the constructive suggestions.
Referee #2 (Remarks for Author): In this manuscript, the authors claim that CAR-BCMA T cells are well-tolerated and highly effective in treating refractory MG, resulting in an improvement in physical function and serologic remission.Patient MG-1, with anti-AChR-IgG and anti-Titin antibodies, and patient MG-2, with anti-MuSK-IgG4, showed a poor response to first-line immunosuppressive therapies.Although B-cell-targeting therapy rituximab was considered an early therapeutic option, it also failed in these two patients.The present case series illustrates the potential of CAR-T cell therapy in treating seropositive MG, as evidenced by sustained depletion of autoantibodies and prolonged therapeutic efficacy beyond one year.Response: Thanks again to the reviewer for the encouragement of the highlights of our study.We will revise and improve it point-by-point based on your valuable comments.
The reviewer has concerns regarding the choice of applying CAR T-cell therapy to these patients, as there are other clear options available with lower potential side effects, specifically targeting plasma cells and the effector functions of anti-AChR antibodies.Refer to Mané-Damas, Marina et al. "Novel treatment strategies for acetylcholine receptor antibody-positive myasthenia gravis and related disorders" (Autoimmunity Reviews, vol. 21,7, 2022, 103104. doi:10.1016/j.autrev.2022.103104).Response: Thanks to the reviewer's suggestions.We have thoroughly read this article on the progress of MG treatment and cited it as an important reference (Mané-Damas et al, 2022) (Ref.27).Indeed, there are currently increasing innovative strategies for the treatment of MG were under evaluation in various clinical trials.And novel strategies like complement inhibitors and FcRn antagonists were approved by FDA since 2017.However, minimal options are officially approved and commercially available treatments in mainland China before 2023.Our two patients were enrolled and received CAR-T treatment in the early 2022.At that time, there was only rituximab as off-label choice for refractory myasthenia gravis in China.There are two other teams working on CAR-T in treating MG patients.One performed RNA-engineered anti-BCMA CAR-T treatment in MG patients without lymphodepletion therapy (Granit et al, 2023).This protocol requires repeated administration (twice weekly for 3 weeks, once weekly for 6 weeks, or once monthly for 6 months in Part 2) and has shown maintained improvements during a 5-months follow-up in all participants who received weekly infusions for 6 weeks.Another case report recently published on Lancet Neuro was an anti-CD19 CAR-T treatment (conventional DNA-engineered) given in one patient early this year (2023) (Haghikia et al, 2023).Clinical efficacy and primary safety were observed in the first 62 days after CAR-T treatment.In our report, both the two patients showed clinical remission for up to 18 months after one dose of CAR-T infusion, which provides a relatively long period of observation and gives many hints on safety and long-term efficacy.We hope that our case report could help neurologists around the world learn more about the possibilities of such treatments in autoimmune diseases.Meanwhile, we have now added a lot of related comments in the discussion (Page 11 Line 406-431 in marked version).Thanks again to the reviewers for their constructive suggestions.
As is well known, CAR T-cell therapy is a type of immunotherapy that involves genetically modifying a patient's T cells to express a receptor targeting cancer cells.Neurological side effects, particularly neurotoxicity or cytokine release syndrome (CRS), can occur as a result of this therapy.The onset and duration of neurological side effects can vary widely among individuals.Neurological side effects from CAR T-cell therapy typically manifest within the first few days to weeks after treatment initiation.In some cases, they can occur within hours after the infusion of modified T cells.The duration of these side effects also varies.Some individuals may experience transient and mild symptoms, while others may face more severe and prolonged effects.The severity and timing of neurological side effects are often unpredictable.Symptoms may include confusion, delirium, seizures, and other neurological manifestations.Patients receiving CAR T-cell therapy should have regular follow-up appointments with their healthcare team to monitor for any delayed or long-term side effects.Response: We thank the reviewer for pointing out this issue.According to the available clinical trials of CAR-T therapy on autoimmune diseases and hematological malignancies, most adverse effects occurred in the early stage.To date, no prolonged or delayed adverse events have been reported in the treatment of autoimmune diseases (Mackensen et al, 2022;Muller et al, 2023;Qin et al, 2023).While in hematological tumors, several cases of prolonged toxicity did appear.For instance, two patients with multiple myeloma had developed a grade 3 cerebellar disorder that was considered to be a dose-limiting-toxicity effect for the dose of 450×10 6 CAR-T-anti-GPRC5D at 6.5 and 8.4 months, respectively (Mailankody et al, 2022).Another clinical trial using anti-BCMA CAR-T cells, the patient developed movement disorder with features of parkinsonism at day 101 after CAR-T cell infusion (Van Oekelen et al, 2021).These two neurological adverse events were both speculated to be related with target antigens expressing in the brain tissue.Additionally, prolonged grade 3/4 cytopenia such as neutropenia, thrombocytopenia, anemia and lymphocytopenia, were also reported in several patients, with the susceptibility to develop fatal infectious and hemorrhagic events (Benjamin et al, 2020;Mei et al, 2021;Munshi et al, 2021).In our study, we also pay a lot of attention on side effects of CAR-T therapy, including CRS, ICANS, neurotoxicity, and etc.Fortunately, following treatment with CAR-T cells, patient MG-1 experienced transient grade 1 CRS (pyrexia with maximum temperature 39.3 °C) at day 8, which resolved within 1 day automatically.Patient MG-2 experienced no CRS.Neutropenia and lymphocytopenia were observed within 1-month post-infusion, and all resolved within 4 weeks.
No immune effector cell-associated neurotoxicity syndrome, other neurologic toxic effects, or dose-limiting-toxicity were observed.All AEs were documented, and regular follow-up were performed every 3 months.Currently, both patients have been followed up for more than 18 months and no delayed adverse effects were observed.Nonetheless, subsequent regular follow-up and continuous monitoring are still needed for the prevention of any potential delayed or long-term side effects.We have also added the subsequent follow-up information to the revised Figure 1-2.As suggested, a detailed discussion of the potential side effects that CAR-T therapy may bring and the subsequent monitoring of delayed or long-term side effects has now been added to the Discussion section (Page 11-12 Line 432-457 in marked version).Thank you again for the professional advice.
Revised Figure 1 Revised Figure 2 It is crucial to consider if the potential side effects of this treatment are worthwhile, especially in neurological diseases like MG, when other treatment options are available in the market.I would not consider such a complex approach, as depicted here, for the treatment of refractory MG patients a good current option.Instead, I would seek, if possible, more specific patient-tailored strategies, which will minimize the considerable risk of side effects, especially in the long term.Response: Thanks again to the reviewer for these professional concerns.As doctors specializing in neuroimmunology, we hope that more treatment options could be explored for patients with refractory myasthenia gravis and subsequent frustration.At the same time, we also want to point out that the sustained clinical efficacy for over 18 months following one single dose of CAR-T cell infusion is really exciting.Both the patients really enjoy their drug-free life (all regular administration of oral steroids and all kinds of immunosuppressants were stopped before CAR-T infusion).
For now, the exploration of CAR-T in treating autoimmune diseases is evolving in various research centers around the world (Schett et al, 2023).In addition to the promising clinical effects of CAR-T in treating MG, our study also explored the molecular mechanism of CAR-T cells in treating autoimmune diseases through single-cell transcriptome analysis.Different characteristics of CAR-T cells in autoimmune diseases and those cells in hematological tumors were depicted.It is hoped that through the description of these molecular characteristics, our findings could provide some evidence for more specific patient-tailored CAR-T strategies in treating patients with autoimmune diseases in the future.In addition, CAR-T therapy with more specific patient-tailored strategies, such as MuSK-CAAR T cell technology (Oh et al, 2023), and NMDAR-CAAR T cell therapy (Reincke et al, 2023), were under evaluation in several preclinical studies, while more clinical evidence is warranted.We have now added related comments in the discussion (Page 12 Line 456-457 in marked version).Thank you again for all the professional advices and constructive help.
Referee #3 (Remarks for Author): In this manuscript, Tian and colleagues reported very favorable outcome using chimeric antigen receptor (CAR) T cells targeting BCMA in two patients with refractory myasthenia gravis (one with AChR Ab, and one with MuSk Ab).They also utilized single cell RNA and TCR sequencing to track the temporal evolution of CAR-T phenotypes, and found a shift from autologous T effector (Te) to proliferating cytotoxic-like CD8 clones and NK-like Te cells post treatment in vivo.The clinical outcome and decline in antibody titers at 12 mo are significant and expected, but whether long-term remission will be induced remains to be determined.The work is exciting and very thorough and provides insight into immunologic events occurring after CAR-T cell therapy targeting BCMA in autoimmunity.Data presentation is excellent though may be too crowded /complex in the last 2 figures.Data interpretations are logical.Here are some questions /comments: Response: We appreciate the reviewer's affirmation and suggestions for our study, which guided us to revise this manuscript and strengthen our conclusion.Thank you for pointing out the issue about the data presentation.We've divided Moreover, we also treasured your following construction suggestions, and we've provided point-to-point response as shown below.
Revised Figure 4 Revised Figure 5 Revised Figure 6 1. Cyclophosphamide has occasionally been used in refractory MG.Can the authors comment on the possibility that lymphodepletion prior to CAR-T cell infusion may have partially contributed to favorable outcome?Response: We thank the reviewer for pointing out this issue.Indeed, cyclophosphamide has occasionally been used in refractory MG, which may attribute partially to clinical efficacy we observed in these two patients.In addition, lymphodepletion therapy we adopted in this study include the use of fludarabine, which was not used in treating MG.We cannot exclude the effects of cyclophosphamide and fludarabine, while the effects were supposed not to persist over 3 months.At the same time, both patients have been followed up for more than 18 months.We have also expanded the clinical follow-up data to 18 months and added it to the results and Figures 1-2 (Page 7 Line 247-250 in marked version).In our 18-month follow-up observation, we found that the two patients still showed sustained clinical efficacy.This is inspiring.We have now added related comments as limitations in the discussion (Page 13 Line 515-518 in marked version).Thank you again for all the constructive suggestions.
Revised Figure 1 Revised Figure 2 2. Since the number of targeted cells (BCMA+ B cells) in autoimmune diseases is much lower than in lymphomas or other cancers, is lymphodepletion allowing favorable condition for CAR-T cell proliferation necessary?Response: Thank you to the reviewer for raising such a question worthy of deep consideration.We had similar doubts before designing the trial protocol.However, through literature search, we found that CAR-T cell therapy in some tumors has obvious limitations in its expansion without lymphodepletion (Geyer et al, 2019;Hirayama et al, 2019;Narayan et al, 2022;Turtle et al, 2016).We also noticed that, in a recent report (Granit et al., 2023), RNA-engineered CAR-T cells were administered to MG patients without lymphodepletion chemotherapy.But at the same time, patients were given repeated infusions of CAR-T cells (twice weekly for 3 weeks, once weekly for 6 weeks, or once monthly for 6 months in Part 2).In addition, preclinical studies on CAR-T treating autoimmune diseases also adopted lymphodepletion treatment for allowing favorable condition for CAR-T cells (Jin et al, 2021;Kansal et al, 2019) while CAR-CTLs injected without preconditioning lymphodepletion were barely detectable by day 15 in any of the analyzed tissues (Kobayashi et al, 2020).Collectively, lymphodepletion might be necessary for CAR-T cell treatment, benefiting the homeostasis and expansion of transferred CAR-T cells.Therefore, we conducted lymphodepletion before CAR-T treatment to assure CAR-T cells' successfully expanding.We have added related comments in the revised discussion (Page 11 Line 421-424 in marked version).Thanks again for all the constructive suggestions.

Can authors explain why do CAR-T cells persist only 30d in autoimmune disease compared to longer persistence in cancers?
Response: We totally agreed with the reviewer.The shorter persistence of CAR-T cells (approximately 30 days) in autoimmune diseases compared to that in hematological cancers, was observed in early reports (Mackensen et al., 2022;Qin et al., 2023) and the present study.Unlike cancers, circulating B cells/plasma cells are easily and rapidly cleared, resulting in limited stimulation of functional effector T cells that persist after targeting cell eradication.In addition, our study using single-cell transcriptomic analysis highlights the importance of infusing proliferating CAR + Te cells manufactured from endogenous Te phenotype in the final expansion stage of autoimmunity.The suppressed effector signature and profound mitochondrial dysfunction of Te cells in patients with refractory MG, which might result from multiple/long-term immunosuppressants and steroid use prior to enrolment, and subsequent compromised properties of the manufactured CAR-T cells, provide a possible explanation for their poor persistence in autoimmune diseases, in addition to the relatively low levels of antigen stimulation compared with malignancies.Additionally, inhibited proliferating properties and enhanced cell exhaustion/ dysfunction were also observed in basal cells and manufactured CAR-T cells of MG patients, which might also lead to their relatively shorter persistence and poorer effectiveness, in addition to the limited antigen exposure, enriched cell types, and suppressed effector and profound mitochondrial dysfunction.We have added related comments in the revised discussion (Page 13 Line 502-511 in marked version).1st Revision -Editorial Decision 19th Jan 2024 Dear Prof. Wang, Thank you for the submission of your revised manuscript to EMBO Molecular Medicine.We have now heard back from the one referee who agreed to evaluate your manuscript.This referee also assessed author responses to concerns raised by other referees.I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) We note that you currently have, a total of 3 first authors.Is that correct?Do you confirm equal contribution of these authors, able to take full responsibility for the paper and its content?While there is no limit per se to the number of first authors, 3 authors is rather rare, and may not reflect as intended to the community.-In M&M, add a statistical paragraph that should reflect all information that you have filled in the Authors Checklist, especially regarding randomization, blinding, replication.
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You are also welcome to suggest a striking image or visual abstract to illustrate your article.If you do please provide a jpeg file 550 px-wide x 300-800px high.10) A Conflict of Interest statement should be provided in the main text 11) Please note that we now mandate that all corresponding authors list an ORCID digital identifier.This takes <90 seconds to complete.We encourage all authors to supply an ORCID identifier, which will be linked to their name for unambiguous name identification.
Currently, our records indicate that there is no ORCID associated with your account.We are pleased to inform you that your manuscript is accepted for publication and is now being sent to our publisher to be included in the next available issue of EMBO Molecular Medicine.Your manuscript will be processed for publication by EMBO Press.It will be copy edited and you will receive page proofs prior to publication.Please note that you will be contacted by Springer Nature Author Services to complete licensing and payment information.
You may qualify for financial assistance for your publication charges -either via a Springer Nature fully open access agreement or an EMBO initiative.Check your eligibility: https://www.embopress.org/page/journal/17574684/authorguide#chargesguideShould you be planning a Press Release on your article, please get in contact with embo_production@springernature.com as early as possible in order to coordinate publication and release dates.
If you have any questions, please do not hesitate to contact the Editorial Office.Thank you for your contribution to EMBO Molecular Medicine.

EMBO Press Author Checklist USEFUL LINKS FOR COMPLETING THIS FORM
The EMBO Journal -Author Guidelines EMBO Reports -Author Guidelines Molecular Systems Biology -Author Guidelines EMBO Molecular Medicine -Author Guidelines Please note that a copy of this checklist will be published alongside your article.

Abridged guidelines for figures 1. Data
The data shown in figures should satisfy the following conditions: New materials and reagents need to be available; do any restrictions apply?Yes Data Availability

Antibodies
Information included in the manuscript?
In which section is the information available?
(Reagents and Tools For antibodies provide the following information: -Commercial antibodies: RRID (if possible) or supplier name, catalogue number and or/clone number -Non-commercial: RRID or citation

DNA and RNA sequences Information included in the manuscript?
In which section is the information available?
(Reagents and Tools

Core facilities Information included in the manuscript?
In which section is the information available?
(Reagents and Tools

Sample definition and in-laboratory replication
Information included in the manuscript?
In which section is the information available?
(Reagents and Tools Studies involving human participants: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.

Materials and Methods
Studies involving human participants: Include a statement confirming that informed consent was obtained from all subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.

Materials and Methods
Studies involving human participants: For publication of patient photos, include a statement confirming that consent to publish was obtained.

Reporting
Adherence to community standards Information included in the manuscript?
In which section is the information available?
(Reagents and Tools Have primary datasets been deposited according to the journal's guidelines (see 'Data Deposition' section) and the respective accession numbers provided in the Data Availability Section?

Data Availability
Were human clinical and genomic datasets deposited in a public accesscontrolled repository in accordance to ethical obligations to the patients and to the applicable consent agreement?

Data Availability
Are computational models that are central and integral to a study available without restrictions in a machine-readable form?Were the relevant accession numbers or links provided?

Not Applicable
If publicly available data were reused, provide the respective data citations in the reference list.

Yes Results
The MDAR framework recommends adoption of discipline-specific guidelines, established and endorsed through community initiatives.Journals have their own policy about requiring specific guidelines and recommendations to complement MDAR.
10) We replaced Supplementary Information with Expanded View (EV) Figures and Tables that are collapsible/expandable online.A maximum of 5 EV Figures can be typeset.EV Figures should be cited as 'Figure EV1, Figure EV2" etc... in the text and their respective legends should be included in the main text after the legends of regular figures.
Figures 1-2 (Page 7 Line 247-250 in marked version).Within one and a half years after stopping steroids and immunosuppressants, both patients showed sustained clinical efficacy in relieving clinical symptoms.The sustained remission should have slight effects from previous treatments.We have written in the limitations (Page 13 Line 515-518 in marked version) the possible short-term effects of previous treatments.Thanks again for the reminders and help.
2) Formatting: Please correct order of the manuscript sections: Abstract / Introduction / Results / Discussion / Materials and Methods / Data Availability / Acknowledgements / Disclosure and competing interests statement / The Paper Explained / For More Information / References / Figure legends / Tables and their legends / Expanded View Figure legends 3) In the main manuscript file, please do the following: -Please address all comments suggested by our data editors listed below: o Figure legends: 1. Please indicate the statistical test used for data analysis in the legends of figures 3f; 6b-c.2. Please note that the box plot needs to be defined in terms of minima, maxima, centre, bounds of box and whiskers, and percentile in the legend of figure 3e. 3. Please note that information related to n is missing in the legends of figures 3e; 6b-c; EV 4a-b.-Updated the callouts of EV figures in the text to Fig EV1-4.-In M&M, provide the statement that the experiments conformed to the WMA Declaration of Helsinki and to the principles set out in the Department of Health and Human Services Belmont Report.
10) Please provide a point-by-point letter INCLUDING my comments as well as the reviewer's reports and your detailed responses (as Word file).I look forward to reading a new revised version of your manuscript as soon as possible.Instructions to submit your revised manuscript *** *** PLEASE NOTE *** As part of the EMBO Publications transparent editorial process initiative (see our Editorial at https://www.embopress.org/doi/pdf/10.1002/emmm.201000094),EMBO Molecular Medicine will publish online a Review Process File to accompany accepted manuscripts.
1) a .docxformatted version of the manuscript text (including Figure legends and tables) 2) Separate figure files* Figures are not edited by the production team.All lettering should be the same size and style; figure panels should be indicated by capital letters (A, B, C etc).Gridlines are not allowed except for log plots.Figures should be numbered in the order of their appearance in the text with Arabic numerals.Each Figure must have a separate legend and a caption is needed for each panel.*Additional important information regarding figures and illustrations can be found at https://bit.ly/EMBOPressFigurePreparationGuideline.See also figure legend preparation guidelines: https://www.embopress.org/page/journal/17574684/authorguide#figureformat

In which section is the information available?
definitions of statistical methods and measures: (Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section)

Table ,
Materials and Methods, Figures, Data Availability Section)

materials Information included in the manuscript? In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)Cell lines: Provide species information, strain.Provide accession number in repository OR supplier name, catalog number, clone number, and/OR RRID.

In which section is the information available?
Provide species, strain, sex, age, genetic modification status.Provide accession number in repository OR supplier name, catalog number, clone number, OR RRID.
(Reagents and ToolsTable, Materials and Methods, Figures, Data Availability Section) Laboratory animals or Model organisms:

In which section is the information available?
(Reagents and ToolsTable, Materials and Methods, Figures, Data Availability Section) If collected and within the bounds of privacy constraints report on age, sex and gender or ethnicity for all study participants.Yes Materials and Methods & Results

In which section is the information available?
Table, Materials and Methods, Figures, Data Availability Section) (Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

In which section is the information available?
Table, Materials and Methods, Figures, Data Availability Section)In the figure legends: state number of times the experiment was replicated in laboratory.
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

Use Research of Concern (DURC) Information included in the manuscript? In which section is the information available?
Not Applicable Studies involving experimental animals: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.Include a statement of compliance with ethical regulations.(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) Could your study fall under dual use research restrictions?Please check biosecurity documents and list of select agents and toxins (CDC): https://www.selectagents.gov/sat/list.htmNot Applicable If you used a select agent, is the security level of the lab appropriate and reported in the manuscript?Not Applicable If a study is subject to dual use research of concern regulations, is the name of the authority

granting approval and reference number for
the regulatory approval provided in the manuscript?

and III randomized controlled trials
Table, Materials and Methods, Figures, Data Availability Section) State if relevant guidelines or checklists (e.g., ICMJE, MIBBI, ARRIVE, PRISMA) have been followed or provided.Not Applicable For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right).See author guidelines, under 'Reporting Guidelines'.Please confirm you have followed these guidelines., please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission.See author guidelines, under 'Reporting Guidelines'.Please confirm you have submitted this list.Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) (